We have recently established 65 clonal strains of pituitary anlage cells derived originally from Rathke's pouch epithelial cells. We determined by radioimmunoassay that one of these clones (2A8 clone) secreted simple protein hormones (ACTH, GH and prolactin) but no glycoprotein hormones (TSH, LH and FSH) in culture. However, when the 2A8 clonal cells were transplanted under the kidney capsule or into the hypothalamus of hypophysectomized rats a clear cytodifferentiation of gonadotrophs and thyrotrophs was recognized. We will return these gonadotrophs to in vitro culture and then use these modified 2A8 clonal cells, which are known to contain differentiated gonadotrophs, as starting material for establishment of clones and subclones of gonadotrophs by means of our sepharose-bead separation technique. The subclones will be separately established by different responses to estrogen or progesterone supplemented into culture media. We propose to carry out the baseline study of clonal gonadotrophic cells and their subclones using routine light microscopy, immunocytochemistry, transmission and scanning electron microscopy and radioimmunoassays. In addition, biological activities of clonal gonadotrophic cells and the subclones will be measured by physiological and morphological examination of target organs of hypophysectomized rats after the cells are implanted into the hypothalamus.